Studies were performed to determine whether H-2 restricted anti-TNP cytotoxic T cells recognize TNP covalently coupled to cell membrane H-2 molecules. BY exposing cells to varying concentrations of TNBS, we prepare target cells that did or did not have their H-2 molecules derivatized with TNP. We found that only those target cells having TNP covalently coupled to H-2 were sensitive to lysis by anti-TNP cytotoxic effector cells. Target cells that had TNP covalently coupled to H-2 were placed in culture. After 24 hours, these cells no longer had TNP-H-2, but did express TNP and underivatized H-2 molecules. When they were tested for sensitivity to lysis by anti-TNP cytotoxic T cells, it was found that the amount of lysis was greatly reduced compared to control target cells. Thus, this data is in agreement with the hypothesis that the primary specificity of anti-TNP cytotoxic T cells is directed against TNP molecules covalently coupled to H-2. Studies were undertaken to determine the frequency of cytotoxic T lymphocyte precursors (TTL.P) in H-2d mutant strains sensitized against the wild-type strain. When a mutant of H-2D (strain M504 (H-2Dda) was sensitized against the strain of origin (B10.D2), the CTL. frequency, as determined by limiting dilution analysis, was comparable to that observed between 2 strains that had different alleles at H-2D. This indicates that the relative number of CTL.P that can be sensitized between mutant and strain of origin animals is as great as that observed in conventional H-2 allogeneic responses. Cytotoxic effector cells were generated against an antigen associated with or controlled by the Qa-2 locus by sensitizing BALB/By mice with BALB/cJ lymphoid cells. The effector cells were H-2 unrestricted and required in vivo priming prior to in vitro challenge. Thus, this study establishes that in addition to H-2K, IA, and D, there are additional antigen(s) that can be recognized by cytotoxic T cells, and these antigens are controlled by genes that map in the H-2D-T1a interval.